| The estrogen receptor (ER) is a ligand inducible transcription factor that regulates many physiological processes. ER has two subtypes, ERα and ERβ, and is a member of the nuclear receptor superfamily. Nuclear receptors interact with coregulator proteins that modulate their transcriptional activity. I have characterized a novel 97-kilodalton DEAD box RNA helicase (DP97) that functions as a nuclear receptor coregulator. I have also developed a method to examine the dynamic interactions of either ERα or ERβ with coregulator proteins in cells.; I have identified the DP97 gene and shown that the encoded protein has biochemical ATPase activity. I have also shown that DP97 interacts with ER and with other nuclear receptors both in vitro and in mammalian cells. Also, in reporter gene assays of transiently transfected cells, DP97 represses the transcriptional activity of the agonist-occupied nuclear receptors, but not that of the transcriptional activators p53 or VP16.; I have shown that the N-terminal helicase region of DP97 is dispensable for its activity as a transcriptional coregulator. I also analyzed truncated DP97 proteins for their intrinsic ability to repress a constitutively active reporter gene construct. This led to my finding a region of DP97 that has intrinsic repression activity. This region of DP97 has significant homology with a repression domain in the SMRTe/NCoR2 corepressor. Therefore, DP97 functions as an RNA helicase, a nuclear receptor interacting protein, and a transcriptional coregulator.; ER is able to alter large-scale chromatin structure. I have been able to demonstrate that ERβ, as a well as ERα, is able to alter the structure of gene-amplified chromosome arms containing large numbers of lac operator sites. Using this system, I also examined the recruitment of different coregulator proteins to either ERα or ERβ. I have investigated the transcriptional activity of ERα and ERβ utilizing subtype selective ligands that could preferentially recruit different coregulator proteins.; This thesis reports studies in which I have characterized a novel DEAD box RNA helicase as a nuclear receptor coregulator. I have also examined the similarities in the actions of ERα and ERβ using large-scale chromatin arrays and ER subtype-selective ligands. |
